Spheresomes are the main extracellular vesicles in low-grade gliomas

Cancer progression and its impact on treatment response and prognosis is deeply regulated by tumour microenvironment (TME). Cancer cells are in constant communication and modulate TME through several mechanisms, including transfer of tumour-promoting cargos through extracellular vesicles (EVs) or oncogenic signal detection by primary cilia. Spheresomes are a specific EV that arise from rough endoplasmic reticulum–Golgi vesicles. They accumulate beneath cell membrane and are released to the extracellular medium through multivesicular spheres. This study describes spheresomes in low-grade gliomas using electron microscopy. We found that spheresomes are more frequent than exosomes in these tumours and can cross the blood–brain barrier. Moreover, the distinct biogenesis processes of these EVs result in unique cargo profiles, suggesting different functional roles. We also identified primary cilia in these tumours. These findings collectively contribute to our understanding of glioma progression and metastasis.

Additionally, cells receive signals from the microenvironment via primary cilium 53,54 , a microtubule-based subcellular structure that protrudes from the cell and plays different roles in cellular processes, including tumoural progression 53,54 . Moser et al. 43 reported that the presence of primary cilia in high-grade gliomas correlates with higher rates of tumour proliferation, showing aberrant ciliogenesis in these tumours. Conversely, Sarkisian et al. 44 suggest the presence of the primary cilium in glioblastomas. Moreover, recent compelling studies indicate that primary cilium also emits signals through the release of EVs 23,55-57 . EVs are involved in tumour biology, including malignization of distant cells. In addition, they show a potential role in the non-invasive diagnosis of brain tumours. To our knowledge this work provides the first evidence of spheresomes genesis and activity in low-grade human gliomas using electron microscopy. Furthermore, primary cilia biogenesis and its relation with EVs are also depicted.

Methods
A more detailed version of the Methods can be found as Supplementary Information. Human samples. Human biopsies were obtained from surgical resections collected in the Department of Pathology at the University Clinic Hospital of Zaragoza, Spain. Biopsy samples from 5 patients fulfilling histological criteria of low-grade glioma were used in this study. Patients were all males of Spanish origin with a mean age of 63 (± 6.9) years. All procedures were approved by the Human Research Ethics Committee (Comité Ético Figure 1. EVs biogenesis. Exosomes originate via the clathrin-mediated endocytic pathway. Cell membrane endocytosis results in the formation of early endosomes that develop late endosomes. Late endosomes' membrane invaginates creating intraluminal vesicles (ILVs), originating multivesicular bodies (MVB). When MVB fuses with the plasma membrane, exosomes (originated from ILVs) are released. Conversly, ectosomes originate from direct budding from plasma membrane. Proteins with lipid anchors (garnet dots) accumulate and help curving the membrane as the bulge increases. Biomolecules accumulate within the bulges until they are cleaved and released as ectosomes. Spheresomes biogenesis begins via RER-Golgi-derived vesicles which aggregate beneath the cell membrane. As the number of vesicles increases, the cell membrane evaginates towards the extracellular space, originating a multivesicular sphere (MVS) that is subsequently released to the extracellular medium. MVS membrane rupture releases spherosomes. Membrane proteins (exosomes and ectosomes): pink and purples; RER-Golgi proteins (spheresomes): black and blue.

Transmission electron microscopy (TEM).
Tumour samples (about 1-1.5 mm 3 ) were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde overnight at RT, washed in 0.1 M phosphate buffer for 5 min, postfixed with 2% osmium, rinsed, dehydrated in graded acetone (30%, 50%, 70% with 2% uranyl acetate, 90%, 100%), cleared in propylene oxide and embedded in araldite (Durcupan, Fluka AG; Buchs SG, Switzerland). Semi-thin Sections (1.5 μm) were cut with a diamond knife, lightly stained with 1% toluidine blue and examined by light microscopy (Olympus BX51 microscope, Olympus Imaging Corporation; Tokyo, Japan). Later, ultrathin (0.05 μm) sections were cut, collected on Formvar-coated single-slot grids counterstained with 1% uranyl acetate and Reynold's lead citrate. The sections were examined under a FEI Tecnai G2 Spirit TEM. The images were captured with Advanced Microscopy Techniques, using a Corp. Charge-Coupled Device imaging system (CCD from Danvers, MA, USA). The number of MVS and MVB was counted in micrographs of 64 cells containing one or both of these vesicular structures. Within each type of MV the number of vesicles was also counted. In addition, the average diameter of MVS (number of MVS assessed, n = 30), MVB (n = 5) and of the vesicles within them (spheresomes n = 100; ILV or exosomes n = 50) was calculated using FIJI ImageJ software 58 .

Results
Histological and immunohistochemical features of low-grade glioma. Conventional H&E study of the low-grade gliomas revealed numerous fusiform cells with a predominant fibrillar component and significant vascularisation composed by normal endothelial cells ( Fig. 2A). GFAP staining confirmed an astroglial composition ( Fig. 2B) while Ki67 immunostaining showed a moderate rate of mitosis (Fig. 2C). These findings confirm the diagnosis of low-grade glioma, likely a diffuse astrocytoma (WHO grade 2). Primary cilia presence was identified by co-location of acetylated beta tubulin (staining axoneme microtubules) and pericentrin (specific of centrioles and basal bodies) (Fig. 2D).

Ultrastructural features of low-grade glioma. Tumour cells of low-grade glioma exhibit mild nuclear
pleomorphism with oval nuclei that have smooth contours and a narrow band of marginal chromatin. No conspicuous nucleoli were observed, but some tumoural cells displayed nuclear envelope-limited chromatin sheets (ELCS) (Fig. 2E), which are fine nuclear envelope prolongations containing chromatin that have been previously described in other tumours. The cytoplasm of these cells contains abundant granular endoplasmic reticulum related to Golgi dyctiosomes, polyribosomes and mitochondria. Cancer cells established small contacts through cytoplasmic prolongations, composed by parallel bundles of gliofilaments (Fig. 2F). This bundles acquire sometimes an abnormal concentric disposition (Fig. 2G). Moreover, multivesicular bodies (MVB) containing intraluminal vesicles (ILV)/exosomes and MVS containing spheresomes were frequently observed (Fig. 2H). Other types of EVs (i.e. ectosomes or microvesicles) were not observed.

Morphometry of EVs in low-grade gliomas.
In the present study a total on 49 multivesicular spheres

Migration of MVSs.
MVSs released from the tumoural glioma cells can transport cargos both locally and distantly. Locally, MVSs reach neighboring cells in the tumour microenvironment, frequently through prolongations from one or more of these nearby cells (Fig. 3F). Spherosomes are contained in MVSs through the extracellular space and released upon rupture of the MVS membrane (Fig. 3G). Furthermore, MVSs might also reach distant tissues, mainly through blood vessels, as evidenced by the presence of these structures in vessels lumen (Fig. 3H,I).
Presence and biogenesis of primary cilia. Primary cilia were found in low-grade gliomas, and electron microscopy was used to track ciliogenesis process in these tumoural cells. Ciliogenesis begins with the activation of one of the centrioles, the so-called mother centriole (Fig. 4A). Centriole activation is marked by the formation of subdistal appendages, cargo traffic into the centriole, and accumulation of Golgi-derived vesicles near the distal pole. These vesicles fuse to form a big ciliary vesicle (which will form the ciliary membrane) that anchors to the mother centriole through transition fibers. Then, 9 + 0 primary cilia axoneme starts to grow protruding in  www.nature.com/scientificreports/ the ciliary vesicle ( Fig. 4B-C). Finally, ciliary vesicle binds the plasma membrane and the full-length axoneme is exposed to the extracellular medium (Fig. 4D). As depicted in Fig. 4E-F, primary cilia show structural integrity and is usually located in the perinuclear region. Notably, Fig. 4F shows the presence of small extracellular vesicles located in a region of the ciliary pocket membrane with an apparent clathrin-coated pit.

Discussion
How glioma cells influence tumour microenvironment (TME) to promote progression has raised important attention 22 . Communication between tumoural cells and TME can occur through different pathways 25 , including tumour-derived EVs 59-61 . Our findings reveal that glioma tumoural cells frequently produce EVs, including multivesicular spheres (MVSs) and spheresomes, in human low-grade gliomas (Fig. 5).
Exosomes are the best-studied type of EV 62 , while spheresomes have often been misidentified as other structures, such as endocytic vesicles 52 or exosomes 63 . However, proper study of the localisation and features of MVSs and spheresomes in various contexts, particularly in tumours, can provide insight into their functions and their potential use as biomarkers or therapeutic targets.
Analysing the distinct features of the different EVs may be crucial to understand the biology of tumours. Exosomes, ectosomes, and spheresomes share a lipid bilayer, similar morphology, and size range, being difficult to differentiate from each other [5][6][7] . Their biogenesis, however, is completely different [1][2][3][4][5][6]8,9 . While exosomes are generated through the endocytic pathway by budding from membrane fusion of multivesicular bodies with the cell membrane; ectosomes carry cytosolic proteins synthetised by cytosolic polyribosomes 64 . In contrast, MVSs gradually accumulate spheresomes, which are vesicles from the RER-Golgi pathway 5 . As a result, each type of EVs regulates the intercellular transport of proteins synthesised via specific pathways. Proteins synthesised by free polyribosomes accumulate as cargo in ectosomes and exosomes, while proteins synthesised in the RER-Golgi www.nature.com/scientificreports/ pathway would be transported by spheresomes. Therefore, the protein composition of EVs is type-specific, suggesting that spheresomes may play a unique role different from exosomes or ectosomes.
In this work we have demonstrated the coexistence of exosomes and spheresomes in human low-grade gliomas. These findings have been previously identified in other tumoural types, like gastrointestinal stromal tumours (GISTs) 5 . Our observations reveal the presence of multivesicular bodies containing exosomes in the cytoplasm of tumoural cells, which are heading towards the cell surface, as well as MVSs surrounding extracellular environment and in blood vessels lumen. In other studies regarding EVs in gliomas, MVSs were often misidentified as groups of endocytic vesicles 52 . In human biopsies, it is difficult to establish whether all tumour cells produce MVBs and MVSs or only a subset of them. However, we have been able to establish that both forms can coexist in the same tumour. Further research is needed to elucidate what specific signals regulate EVs production. Moreover, it would be important to investigate if these EVs are produced at a specific stage of the tumour progression or continuously throughout the tumour´s biology.
Tumour-derived EVs play remarkable roles in facilitating tumoural invasion and metastasis by modulating distant tissues [65][66][67] . In central nervous system, EVs can cross the BBB and travel through the bloodstream 68 . Research on EV cargo from glioblastoma patients has revealed different molecules that can become significant diagnostic and prognostic markers 69 . We have observed the presence of spheresomes within tumour vessels that allow MVSs to migrate to distant organs or tissues. This finding could be of great interest for early non-invasive diagnosis of these tumours. Additionally, exosomes and exosome-mimics have been used as drug delivery carriers in glioblastoma models, showing potential therapeutic applications [70][71][72] .
Primary cilium plays different roles in tumoural development 53,54 . The genesis of certain tumours is related to alterations in specific ciliary membrane proteins that transmit abnormal proliferation signals, including Sonic hedgehog (Shh) pathway. Both presence/increase of primary cilia and their loss have been observed in tumours 73 . The presence/increase of the cilium has been reported both in mesenchymal and epithelial tumours; such as, gastrointestinal stromal tumours 74,75 , giant cell tumours of bone 76 , bladder tumours 77 , meningioma 78 , osteosarcoma 79 , pancreatic adenocarcinomas 80 , lung adenocarcinomas 80 , muscular rabdomiosarcomax 81 , or glioblastoma 44 . The loss of primary cilia has been shown in breast tumours 82,83 , prostate tumours 80,84 , renal tumours 85 , pancreatic adenocarcinomas 86 , colangiocarcinoma 87 , melanoma 88,89 , and glioblastoma cell cultures 90 . www.nature.com/scientificreports/ Additionally, the structural dynamism of the primary cilium allows this organelle to appear/disappear at different times or stages of the disease 91,92 . It has been observed that primary cilium can be present both in premalignant and malignant lesions [93][94][95][96][97][98][99] . Our results support the presence of primary cilia in low-grade gliomas, although this does not exclude the possibility of their presence in other stages of the tumour, depending on the needs of the cells.
Normal astrocytes show primary cilium to promote cell proliferation and differentiation 91 . In high-grade gliomas, Moser et al. 43 reported an aberrant primary ciliogenesis. However, Sarkisian et al. 44 showed opposite results. Consistent with these findings, our results suggest that primary cilium is formed in early stages of astrocyte tumour transformation, serving as an antenna that receives signals from the extracellular environment, without displaying any structural alterations.
Exosomes can also be derived from the primary cilium, as shown in previous studies. 56,57,100 Accordingly, we observed the presence of vesicles in the proximity of ciliary pocket membrane. Masyuk et al. 101 demonstrated for the first time that exosomes can be involved in intercellular communication by interacting with primary cilia, and that ciliary extracellular vesicles contain functional proteins that play a key role in cilia biology. Recent findings have identified that ciliary extracellular vesicles and cytosolic extracellular vesicles have unique and distinct features, highlighting their different properties. 102 Although we cannot establish the exact origin of the vesicles found close to the ciliary membrane, it is possible that they have an autocrine regulatory effect on the tumour cells themselves via the primary cilium sensing.

Data availability
All data generated or analysed during this study are included in this published article and it supplementary information file.